Reference solution

ABSTRACT

A reference solution is provided, which comprises, in a liquid phase, at least one of a first compound and a second compound which are mutually convertible into each other, and at least one catalyst, which catalyzes the conversion between the first compound and the second compound.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Appl. No.60/554,417, filed Mar. 19, 2004 and to Danish Patent Appl. No.PA200301758, filed Nov. 28, 2003, each of which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to a reference solution, a kit forproviding a container holding the reference solution, a method ofoperating the kit, a container holding the reference solution, a methodof preparing the reference solution and use of the reference solution.

BACKGROUND OF THE INVENTION

Sensors for measuring parameters in a test fluid are widely used invarious fields of chemistry, biology and physiology.

In order to assure that sensor measurements are accurate, the sensorshould be regularly calibrated. Calibrating the sensor typicallyinvolves determining experimentally the correspondence between a sensorresponse and a predetermined parameter value of a reference material,and adjusting the sensor in accordance therewith.

The quality of the sensor performance should also be controlled on aregular basis to verify experimentally that the sensor measurements areaccurate. This is usually done by comparing a measured parameter valueof a reference material to an acceptance range of the same referencematerial.

Reference materials for calibration and quality control routinescomprise compounds, which represent the parameter in question. Theparameter may be a physical parameter, such as viscosity, density,pressure and conductivity, or a chemical parameter, such as pH or theconcentration of gasses, electrolytes or metabolites of a physiologicalliquid, like blood. The reference materials should represent theparameter precisely and steadily over their lifetime. Therefore,reference materials may comprise compounds in concentrations which arestable over time, e.g., compounds in chemical equilibrium, and theyshould be prepared and stored under closely controlled conditions toassure fulfillment of their specifications.

In some cases such controlled conditions of preparation and storage maynot suffice to fulfill the specifications. This may be the case with areference solution comprising two compounds which exist in equilibriumand which are mutually convertible into each other. The equilibriumbetween such two compounds may be temperature dependent. If so, then,any temperature change is likely to gradually change the chemicalcomposition of the reference solution.

One such reference solution may include a dissolved gas, in which thegas may be distributed between a gaseous phase in a container headspaceand a dissolved phase in the solution. With such a system, theequilibrium distribution of gas between the two phases may betemperature dependent. Thus, if the solution is stored at a temperaturedifferent from the operating temperature, the distribution of gas may bedifferent from the equilibrium distribution at the operatingtemperature. The reference solution may therefore need long termconditioning before use in order to establish a state of equilibriumbetween the two phases corresponding to the operating temperature.

Another type of two-phase reference solution is a system comprising asparingly soluble compound, which may exist partly as solute and partlyas solid phase. The distribution between these two phases may betemperature dependent, and the solution may also need long termconditioning before use in order to establish a state of equilibrium atthe operating temperature.

Still other reference solutions involve chemical reactions, in which twoor more compounds exist in equilibrium. Examples include the systems ofglutamine and glutamic acid/glutamate and of carbon dioxide and carbonicacid, as well as the mutarotational system of α-D-glucose andβ-D-glucose. Reference solutions comprising these compounds may requireconditioning for days, months or even years before use in order toestablish a state of equilibrium at the operating temperature.

Yet another example of a system comprising two compounds in equilibriumis the system of creatinine and creatine. As with the examples above,long term conditioning may be needed as the equilibrium between thesetwo compounds is established only over a period of months or years.

International Published Patent Application WO 02/14533 to RocheDiagnostics GmbH discloses a method of calibrating a creatinine sensor.According to this method, two calibration solutions are used. The firstcalibration solution is an acidic solution of creatinine, which is firstneutralized before being used for calibration. The neutralization step,however, makes this procedure inconvenient for daily operation. Thesecond solution is a solution of creatinine and creatine at equilibriumconcentrations corresponding to a specific temperature. However, toavoid compositional changes, such a solution should either be preparedimmediately before use or kept at that specific temperature, both ofwhich are impractical in daily use.

Thus, despite the hitherto proposed reference solutions for detection ofmutually convertible compounds, there is still a need for a referencesolution which does not require extended conditioning and/or instantpreparation. Accordingly, it is an object of the present invention toprovide such a reference solution.

SUMMARY OF THE INVENTION

In one aspect of the invention, a reference solution is provided whichcomprises, in a liquid phase, at least one of a first compound and asecond compound, which are mutually convertible into each other, and atleast one catalyst, which catalyzes the conversion between the first andsecond compounds.

Another aspect of the invention is a kit for providing a containerholding the reference solution of the invention, comprising a containerholding the reference solution described above, comprising at least afirst compartment and a second separate compartment; a liquid firstphase contained in the first compartment; and a second phase containedin the second separate compartment, said second phase comprising the atleast one of a first compound and a second compound and the at least onecatalyst.

In another aspect of the invention, a method of operating the kit isprovided.

In another aspect of the invention, a container holding the referencesolution is provided.

In another aspect of the invention, a method of preparing the referencesolution of the invention is provided, comprising the steps of adding toa liquid phase at least one of a first compound and a second compound;and at least one catalyst; and determining, before or after addition ofthe at least one catalyst, the equilibrium concentrations of the firstcompound and the second compound for a selected temperature.

In another aspect of the invention, use of the reference solution of theinvention for calibration or quality control of a sensor is provided.

FIGURES

FIG. 1 shows a kit for providing a container holding a referencesolution according to an exemplary embodiment of the invention.

FIG. 2 shows comparable data for the concentration of creatinine vs.time upon conditioning of reference solutions with and without enzymepresent.

DETAILED DESCRIPTION

As used herein, the term “reference solution” means a solution forcalibration and/or quality control of sensors. Thus, the referencesolution may be used for calibration of a sensor for which it isintended, and/or it may be used for quality control of the sensor.

The term “liquid phase” means the solvent of the reference solution, ore.g., a buffer solution, or a solution of one or more compounds otherthan the mutually convertible compounds for which it may also serve as areference solution.

The term “compound” means a chemical substance or a mixture of chemicalsubstances. According to the invention, the species to be determined bythe sensor may in fact be one of the first and the second compounds ofthe reference solution, or it may be a reaction product derived from thefirst and/or the second compound(s).

The term “catalyst” may refer to a single catalyst, which catalyzes theconversion reaction in both directions, or to a combination of, e.g.,two catalysts, one of which catalyzes the conversion reaction in onedirection and the other of which catalyzes the conversion reaction inthe opposite direction.

As used herein, the term “mutually convertible” means that the compoundsof the reference solution, may, by chemical reaction in eitherdirection, be transformed into one another.

As further used herein, the term “equilibrium concentrations” means theconcentrations of the first and the second compounds in the referencesolution when the reference solution is in a state of equilibrium at agiven temperature, i.e., when there is no net conversion between thefirst and the second compounds at the given temperature.

The term “separate compartment” means a compartment of the describedcontainer which is adapted to hold a phase in such a way, that it is notin contact with any other phases held in any other compartments of thecontainer.

As used herein, the term “computing the equilibrium concentrationtherefrom” means calculating, from the total concentration of the firstand the second compound, the equilibrium concentration of each compoundat a given temperature. The calculation may be based on a predeterminedalgorithm that includes equilibrium data, e.g., equilibrium constants asa function of temperature.

As used herein, the term “computing the equilibrium concentration of theother compound therefrom” means calculating from the equilibriumconcentration of the first compound the equilibrium concentration of thesecond compound at a given temperature. The calculation may be based ona predetermined algorithm that includes equilibrium data, e.g.,equilibrium constants as a function of temperature.

As used herein, the term “predetermined amount(s)” means that the firstand/or second compound(s) are added in (an) amount(s), corresponding to(a) specific concentration(s) of the compound(s), determined prior toaddition to the liquid phase, by e.g., a weighing procedure. Thus, theamounts added may correspond to the equilibrium concentrations of thefirst and second compounds at the operating temperature of the referencesolution, i.e., the reference solution as prepared may be in a state ofequilibrium, or it may correspond to non-equilibrium concentrations. Inthe latter case the equilibrium concentration may be computed as definedabove, i.e., based on an algorithm that includes equilibrium data, e.g.,equilibrium constants as a function of temperature.

As used herein, the term “sensor” means any kind of device, which iscapable of selectively interacting with the chemical species ofinterest, thereby producing a well-defined and measurable response whichis a function of a desired characteristic of that particular chemicalspecies. Relevant types of sensors are those adapted to determine e.g.,any of the previously mentioned parameters, for example potentiometricsensors, where the response appears in the form of an electricalpotential; amperometric sensors, where the response appears in the formof an electrical current; optical sensors; piezoelectric sensors;thermometric sensors; pressure-change sensors; acoustic sensors or anycombination thereof.

As used herein, the term “conditioning” means allowing the referencesolution to adjust to a given temperature.

Compared to traditional reference solutions which are limited to twomutually convertible compounds, the reference solution according to theinvention also contains a catalyst, which catalyzes the conversionbetween the first compound and the second compound. The catalystaccelerates the conversion between the first and the second compounds,ensuring the fast establishment of a state of equilibrium between thetwo compounds at the operating temperature of the reference solution.Thus, the reference solution of the present invention eliminates theneed for extended periods of conditioning and instant preparation. Aftera reduced period of conditioning, the reference solution of the presentinvention is in a state of equilibrium, and its chemical composition canbe determined entirely from its temperature. In this way the referencesolution of the present invention may be easily provided as aready-to-use solution, e.g., as provided in sealed ampoules.

Prior to use, the reference solution may comprise only one compound ofthe first and the second compounds. Thus, even if the solution isprepared from one compound only, i.e., from the first compound only orfrom the second compound only, the state of equilibrium between thefirst and the second compounds is quickly established once the catalystis present and the operating temperature of the reference solution isreached.

The first and second compounds should be mutually convertible into eachother. According to the invention, the first and the second compoundsmay be the same species in two different phases, e.g., gaseous anddissolved carbon dioxide.

The reference solution may comprise one or more catalysts, which aloneor in combination, catalyze the conversion reaction in both directions.The amount of catalyst needed for catalyzing the conversion reactiondepends on the nature of the system. Catalyst degradation, which resultsin some loss of catalytic activity may occur during the lifetime of thereference solution. Therefore, the catalyst concentration should besufficient to catalyze the conversion reaction for the complete lifetimeof the reference solution.

The catalyst may be an enzyme, i.e., a catalyzing protein, or a coenzymenecessary for the action of an enzyme. Alternatively, the catalyst maybe another catalyzing compound, which mimics the effect of an enzyme,e.g., an enzyme-mimicking polymer.

One type of conversion reaction that may be used in the invention is ahydrolysis reaction. Hydrolysis reactions may be catalyzed by e.g.,enzymes that are hydrolases. Thus, in one embodiment of the invention,the catalyst is a hydrolase enzyme.

Amides and esters represent a group of compounds that may be used in theinvention. Thus, in another embodiment of the invention, the firstcompound is an amide or an ester.

Hydrolysis reactions and conversion reactions involving amides or estersare both characterized by their rather low reactions rates, whencompared to more practical periods of conditioning. During long termstorage, however, such reference solutions may still be subject tosignificant compositional changes. In the absence of a catalyst, thereis no establishment of a state of equilibrium at a chosen operatingtemperature within practical time limits. The addition of a catalyst tosuch reference solutions may reduce the conditioning period by orders ofmagnitude.

In reference solutions comprising creatinine-creatine as the first andsecond compounds, the catalyst may be the enzyme creatinineamidohydrolase, EC 3.5.2.10. In this system, the parameter of interestmay be creatinine, creatine or a parameter derived therefrom.

In reference solutions comprising glutamine-glutamic acid/glutamate asthe first and second compounds, the catalyst may be one or more of theenzymes glutaminase, EC 3.5.1.2, and glutamine synthetase, EC 6.3.1.2.In this system, the parameters of interest may be glutamine, glutamicacid or glutamate or a parameter derived therefrom.

In reference solutions comprising carbon dioxide and carbonic acid asthe first and second compounds, the catalyst may be the enzyme carbonicanhydrase, EC 4.2.1.1. In this system, the parameters of interest may becarbon dioxide, carbonic acid or a parameter derived therefrom.

In reference solutions comprising the mutarotational system ofα-D-glucose and β-D-glucose as the first and second compounds, thecatalyst may be the enzyme mutarotase, also referred to as aldose1-epimerase, EC 5.1.3.3. In this system, the parameters of interest maybe either of the two glucose species or a parameter derived therefrom.

The reference solution may comprise additional compounds beyond thefirst and second mutually convertible compounds and the catalyst. Assuch, the reference solution may be used for calibration and/or qualitycontrol of one or more additional sensors for analysis of one or moreadditional parameters. Thus, in addition to being a reference solutionfor a sensor for analysis of the first and/or second compound(s) of e.g.blood, or of a parameter derived therefrom, the reference solution mayalso be used for one or more additional blood parameters such as pO₂,pCO₂, pH, sodium, potassium, calcium, chloride, glucose, lactate, urea,bilirubin, hemoglobin or any derivative of hemoglobin. Such a referencesolution may be used for calibration and/or quality control of aplurality of sensors, thereby reducing the number of required individualreference solutions and saving costs.

A kit for providing a container holding the reference solution of theinvention may comprise a container with at least a first compartment anda second separate compartment, a liquid first phase being contained inthe first compartment, and a second phase being contained in the secondseparate compartment, the second phase comprising at least one of thefirst compound, the second compound and the catalyst of the referencesolution.

The kit of the invention allows for separated storage of the phasesinvolved in the reference solution. With the kit, the reference solutionmay be prepared by combining the separated phases immediately beforeuse, thus extending the lifetime of the catalyst through dry storage.

A first compartment may be used to hold e.g., a buffer solution, and oneor more of the first and second compounds, and the catalyst may be heldin a second separate compartment. Alternatively, a first compartment maybe used to hold a solution of e.g., the first and/or the secondcompound(s), and the catalyst may be held in a second separatecompartment; or a first compartment may be used to hold the catalyst insolution, and the first and/or the second compound(s) may be held in asecond separate compartment.

The first and the second compartments may have any shape whicheffectively separates the phases held in the two compartments. Thus, thesecond compartment may be separated from the first compartment by meansof a thin wall which may be broken or displaced upon the exertion ofmanual pressure on the outer surface of the second compartment, e.g., interms of a blister pack as used in tablet packages. Alternatively, thesecond compartment may be adapted to receive e.g., a plunger, which hasa pointed end. When the plunger is depressed, the end penetrates a thinwall of the second compartment to form a fluid conduit from the secondcompartment into the first compartment, thus delivering the phase fromthe second compartment into the first compartment.

The container may have a third separate compartment, such that at leastone of the first and the second compounds is contained in the secondcompartment and the catalyst is contained in the third compartment.According to this embodiment of the invention, the first and/or thesecond compound(s) are separated from the catalyst during storage. Thefirst compartment may be used to hold a buffer of the referencesolution, while the first and/or the second compound(s) may be held inthe second separate compartment and the catalyst may be held in thethird separate compartment.

The container may comprise additional separate compartments beyond thethree compartments described, e.g., holding compounds other than themutually convertible first and second compounds and the catalyst.

In operating the kit, the second phase may be transferred to the firstphase by providing fluid communication between the compartments. Thus,according to this method, the second phase containing at least one ofthe first and second compounds and the catalyst is transferred to theliquid first phase to prepare the reference solution.

A container holding the reference solution may comprise a firstcompartment and a second separate compartment as described above. Thecontainer may be a two-compartment syringe as is known in the art, or itmay be a two-compartment ampoule.

The reference solution of the present invention may as well be providedin one-compartment containers, e.g., one-compartment ampoules, in whichall of the at least one of the first compound and the second compound,and the at least one catalyst are mixed in the liquid phase.

A method of preparing the reference solution of the invention maycomprise the steps of adding to a liquid phase (a) at least one of thefirst and the second compounds, and (b) at least one catalyst, and,before or after addition of the at least one catalyst, determining theequilibrium concentrations of the first and second compounds for aselected temperature. Steps (a) and (b) may be performed in any order,i.e., step (a) followed by step (b), or step (b) followed by step (a).

The step of determining the equilibrium concentrations of the first andsecond compounds may comprise (a) measuring the total concentration ofthe first and second compounds, and (b) computing the equilibriumconcentrations therefrom.

Determination of the total concentration of the first and the secondcompounds is independent of whether or not the solution is in a state ofequilibrium. Any net conversion, e.g., from the first compound to thesecond compound, will not influence the total concentration of the twocompounds, as an increase in the concentration of the second compoundwill be counterbalanced by a corresponding decrease in the concentrationof the first compound.

In an embodiment of the invention, the step of determining theequilibrium concentrations comprises (a) measuring the concentrations ofeach of the first and second compounds, and (b) computing theequilibrium concentrations therefrom.

In another embodiment of the invention, the step of determining theequilibrium concentrations comprises (a) allowing establishment of theequilibrium concentrations of the first and second compounds afteraddition of the catalyst, (b) measuring the equilibrium concentration ofone of the compounds, and (c) computing the equilibrium concentration ofthe other compound therefrom. In this embodiment of the invention, thereference solution should be in a state of equilibrium, which in turn isobtained upon addition of the catalyst catalyzing the conversion betweenthe first and the second mutually convertible compounds. Uponestablishment of a state of equilibrium, the concentration of one of thefirst or the second compounds is measured.

In yet another embodiment, the step of determining the equilibriumconcentrations comprises the addition to the liquid phase of the firstand/or the second compound(s) in (a) predetermined amount(s), andoptionally computing the equilibrium concentrations therefrom.

The reference solution according to the invention may be used forcalibration or quality control of a sensor, which is sensitive to atleast one of the first and second compounds or a parameter derivedtherefrom. The reference solutions may be stored at a reducedtemperature at which the catalyst degradation is minimized. At thistemperature, the equilibrium between the mutually convertible compoundsmay be shifted compared to their equilibrium at the operatingtemperature of the reference solution.

To establish a state of equilibrium at a conditioning temperature, thereference solution is conditioned in the temperature range of 18-32° C.,preferably 20-25° C., prior to calibration or quality control of thesensor. The conditioning temperature may correspond to the operatingtemperature.

The reference solution may be conditioned for a period in the range of0.1-8 hours, preferably 0.1-3 hours.

Conditioning may be done by leaving the solution at the giventemperature, e.g., at ambient temperature, or by heating or cooling thereference solution by means of a heating or cooling element.Conditioning may also be done on the kit providing the referencesolution. Where the reference solution is stored at a temperature nearthe operating temperature, the need for conditioning may be eliminated.

EXAMPLES

The following describes an embodiment of the invention in which thefirst and the second compounds are creatinine and creatine,respectively, and the catalyst is an enzyme which catalyzes theconversion between creatinine and creatine.

Creatinine, 2-imino-1-methylimidazolidin-4-one, is a degradation productof creatine, α-methyl-guanidoacetic acid, which plays an important rolein the storage and transmission of phosphate-bond energy in the musclesof vertebrates. The compounds are mutually convertible into each other,as shown in (1):Creatinine+H₂O⇄Creatine  (1)

The level of creatinine in blood and urine provides useful informationregarding renal function. Whereas the normal concentration of creatininein blood is in the range 20-80 μM for children and 50-130 μM for adults,concentrations may reach as high as 1500 μM for persons with renalfailure or uremic syndrome.

For the determination of creatinine levels, enzymatic assays based onthe hydrolysis of creatinine to creatine have been developed. In onesuch assay, see e.g., International Published Patent Application WO02/14533, creatinine is converted into creatine, which in turn isconverted to sarcosine, which is oxidized to glycine, formaldehyde andhydrogen peroxide. The concentration of hydrogen peroxide is thendetermined amperometrically. This sensor system is a dual electrodesystem comprising one electrode for the determination of theconcentration of creatine, and another electrode for the determinationof the total concentration of creatinine and creatine. The assay allowsdetermination of the concentration of creatinine from the differencebetween the total concentration of creatinine and creatine and theconcentration of creatine.

The uncatalyzed conversion between creatinine and creatine is slow butsignificant. The rate constant for the first order conversion ofcreatinine into creatine at 25° C. at pH=7.4 is 2.4×10⁻³ d⁻¹. For thereverse reaction it is 1.4×10⁻³ d⁻¹. At 40° C., the rate constants are10.1×10⁻³ d⁻¹ and 8.5×10⁻³ d⁻¹, respectively. At 6° C., the rateconstants are 2.2×10⁴ d⁻¹ and 0.8×10⁻⁴ d⁻¹, respectively. As such, thetime required for uncatalyzed conversion may be counted in days, monthsor even in years.

The catalyzing enzyme for the creatinine-creatine system may becreatinine amidohydrolase, which is also referred to as creatininase.This enzyme, e.g., as supplied by Roche Diagnostics, has an enzymaticactivity of approx. 80 IU/mg, and it may be added to the referencesolution in a concentration in the range of 1-12 IU/ml.

The total concentration of creatinine and creatine in the referencesolution may be in the range of 50-2,000 μM, corresponding to ambienttemperature concentrations of creatinine in the range of approx. 20-750μM and of creatine in the range of approx. 30-1,250 μM. This range ofcreatinine concentrations covers the reference range of children andadults of 20-80 μM and 50-130 μM, respectively, as well the onset levelsof renal insufficiency of approx. 200 μM and of renal failure of approx.500 μM, and thus represents a broad range of practical use.

With reference to the exemplary embodiment shown in FIG. 1, thecontainer 100, made from 1.5 mm polyethylene, for example, comprises twoseparate compartments, 110 and 120. The larger compartment 110 has acapacity of 250 ml and holds 200 ml of a pH=7.4 buffer of HEPES. Thesmaller compartment 120 holds a mixture of 5.7 mg of creatinine, 11.2 mgof creatine and 1500 IU of creatinine amidohydrolase. The smallercompartment 120 is separated from the larger compartment 110 by a 60 μmpolyester membrane 130.

When a moderate manual pressure is exerted on the outer surface of thesmaller compartment 120, the membrane 130 is displaced into the largercompartment 110, and creatinine, creatine and creatinine amidohydrolaseare transferred to the buffer solution in the larger compartment 110.The solution is ready for use after being conditioned for 3 h at theoperating temperature of 25° C. The concentrations of creatinine andcreatine in the reference solution prepared in this manner are 250 μMand 425 μM, respectively.

In a comparison study, three containers, denoted A, B and C, wereprepared as described above, and the concentrations of creatinine andcreatine measured by HPLC. The containers were then stored at 6° C. forone year. Following the one year storage, container (A) was stored foranother 14 days at 6° C., and subsequently conditioned for 3 hours at25° C. Container (B) was stored for another 14 days at 25° C., andsubsequently conditioned for 3 hours at 25° C. The third container (C)was stored for another 14 days at 40° C., and subsequently conditionedfor 3 hours at 25° C.

The concentrations of creatinine and creatine in all three containers A,B and C were determined by HPLC and by a calibrated creatinine/creatinesensor of the type described above. The concentrations of creatinine andcreatine were 250 μM and 425 μM, respectively, at 25° C., i.e., theconcentrations were unchanged compared to the solutions as prepared.

COMPARATIVE EXAMPLE

For comparison, three additional containers, denoted A′, B′ and C′,containing reference solutions with no enzyme, but otherwise identicalto their counterparts A, B and C, were stored and conditioned undersimilar conditions as A, B and C.

Results

With the sample from container A′ (6° C.), the concentrations ofcreatinine and creatine were 243 μM and 432 μM, respectively. With thesample from container B′ (25° C.), the concentrations of creatinine andcreatine were 243 μM and 432 μM, respectively. With the sample fromcontainer C′ (40° C.), the concentrations of creatinine and creatinewere 260 μM and 415 μM, respectively. This comparative studydemonstrates that with solutions containing no enzyme, the relativecreatinine/creatine compositions may deviate significantly from theirequilibrium compositions, i.e., at 25° C.

FIG. 2 shows the creatinine concentration vs. time for samples fromcontainers A and A′ during extended conditioning at 25° C. for a periodof 1 year. During this conditioning period, the creatinineconcentrations of A and A′ were determined at t=0 min., t=15 min., t=60min, t=120 min., t=1,000 min. (approx. 17 hours), t=10,000 min. (approx.7 days), t=100,000 min. (approx. 70 days) and at t=525,600 min. (oneyear) as described above.

At t=0 min., i.e., at the onset of the conditioning period, thecreatinine concentrations of A and A′ were 179 μM and 243 μM,respectively. The concentration of A of 179 μM corresponds to theequilibrium concentration at the storage temperature of 6° C., i.e.,from the onset of the conditioning period, this solution is in a stateof equilibrium. This is not the case with the reference solution of A′,which does not contain a catalyzing enzyme. During the one year storageat 6° C., the creatinine concentration of the solution in A′ was reducedto 243 μM compared to an original concentration of 250 μM. It was,however, still well above the equilibrium level of 179 μM.

At t=120 min., the creatinine concentrations of A and A′ were 250 μM and243 μM, respectively. Thus, at t=120 min., the reference solution of Ahad already reached its state of equilibrium at 25° C., whereas thereference solution of A′ was unchanged compared to the concentration atthe onset of the conditioning period.

At t=525,600 min., i.e., after one year of conditioning, the creatinineconcentrations of A and A′ were 250 μM and 248 μM, respectively. Thus,even after this extended period of conditioning, the reference solutionof A′ had not yet reached a state of equilibrium.

This comparative study demonstrates that the presence of a catalyzingenzyme may significantly reduce the conditioning period required forestablishment of equilibrium, and that in the absence of a catalyzingenzyme, establishment of equilibrium may not be feasible at all.

1. A reference solution comprising, in a liquid phase: at least one of afirst compound and a second compound which are mutually convertible intoeach other; and at least one catalyst, which catalyzes the conversionbetween said first compound and said second compound.
 2. The referencesolution according to claim 1 wherein the at least one catalyst is anenzyme.
 3. The reference solution according to claim 2 wherein theenzyme is a hydrolase.
 4. The reference solution according to claim 1wherein the first compound is an amide or an ester.
 5. The referencesolution according to claim 1 wherein the first compound is creatinineand the second compound is creatine.
 6. The reference solution accordingto claim 5 wherein the at least one catalyst is creatinineamidohydrolase.
 7. The reference solution according to claim 2 whereinthe first compound is carbon dioxide and the enzyme is carbonicanhydrase.
 8. A kit comprising: a container holding the referencesolution according to claim 1, comprising at least a first compartmentand a second separate compartment; a liquid first phase contained in thefirst compartment; and a second phase contained in the second separatecompartment, said second phase comprising the at least one of a firstcompound and a second compound and the at least one catalyst.
 9. The kitaccording to claim 8 wherein the container further comprises a thirdseparate compartment, and wherein the at least one of a first compoundand a second compound is contained in the second separate compartmentand the at least one catalyst is contained in the third separatecompartment.
 10. A method of operating the kit according to claim 8wherein the second phase is transferred to the first phase by providingfluid communication between the compartments.
 11. A container holdingthe reference solution according to claim
 1. 12. A method of preparingthe reference solution according to claim 1, comprising the steps of:adding to a liquid phase: the at least one of a first compound and asecond compound; and the at least one catalyst; and determining, beforeor after addition of the at least one catalyst, the equilibriumconcentrations of the first compound and the second compound for aselected temperature.
 13. The method according to claim 12 wherein thestep of determining the equilibrium concentrations comprises: measuringthe total concentration of the first and second compounds; and computingthe equilibrium concentrations therefrom.
 14. The method according toclaim 12 wherein the step of determining the equilibrium concentrationscomprises: measuring the concentrations of each of the first and secondcompounds; and computing the equilibrium concentrations therefrom. 15.The method according to claim 12 wherein the step of determining theequilibrium concentrations comprises: allowing establishment of theequilibrium concentration of the first and second compounds afteraddition of the at least one catalyst; measuring the equilibriumconcentration of one of the first and second compounds; and computingthe equilibrium concentration of the other of the first or secondcompound therefrom.
 16. The method according to claim 12 wherein thestep of determining the equilibrium concentrations comprises: adding tothe liquid phase the at least one of the first compound and the secondcompound in a predetermined amount; and optionally computing theequilibrium concentrations therefrom.
 17. A method comprising using thereference solution according to claim 1 for calibration or qualitycontrol of a sensor, which is sensitive to at least one of the first andthe second compound or a parameter derived therefrom.
 18. The methodaccording to claim 17 wherein the reference solution, prior tocalibration or quality control of the sensor, is conditioned at aconditioning temperature in the temperature range of 18-32° C.
 19. Themethod according to claim 18 wherein the temperature range is 20-25° C.20. The method according to claim 17 wherein the reference solution,prior to calibration or quality control of the sensor, is conditionedfor a period in the conditioning period range of 0.1-8 hours.
 21. Themethod according to claim 20 wherein the conditioning period range is0.1-3 hours.